Publicação
Development of a novel recombinant subunit antigen as a vaccination target against candidiasis: Cht3 from Candida albicans
| Resumo: | Candida is a genus of ubiquitous yeasts commonly found in healthy individuals and normally acting as harmless commensals in their hosts. However, disruption of the immune defenses can lead to an outgrowth of these yeasts and give rise to local or systemic infections. In fact, Candida, mainly C. albicans, is currently responsible for one of the most common causes of fungal infections worldwide, in part due to an increased development of resistance against the few available therapies. Thus there is an urgent need for the elaboration of new therapies, both for prevention and treatment, among which vaccines may have an important role. Recent studies investigating C. albicans cell wall surface proteins (CWSP) as antigens with DODAB:MO liposomes as carriers and adjuvants led to the detection of a chitinase, Cht3p, as an immune response inducer in mice. Nevertheless, the use of one antigen alone can lead to an evasion of the immune system by C. albicans and hence combination of Cht3p with another antigen, such as the previously studied C. albicans protease Sap2p, is needed. In this study, we successfully developed efficient production and purification protocols for the heterologous production of these two C. albicans proteins. Cht3p could not be produced in Escherichia coli but was successfully produced in a glycosylated and active form in Pichia pastoris. Extracellular production in this host allowed a simplified purification involving ammonium sulfate precipitation and dialysis. Sap2p was successfully produced in both the inactive pro-peptide and active forms in E. coli, with highest production for the former. A simplified, reduced cost, non-chromatographic approach for Sap2p purification, which took advantage of the proteins’ low pH stability, was developed and enabled the obtainment of almost 20 times more purified protein than that previously reported. Preliminary studies investigating various liposomal formulations and conditions previously described and optimized for CWSP (Carneiro et al., 2015) were carried out for both proteins. Cht3p was successfully encapsulated while process optimization is needed for both Sap2p alone and in combination with Cht3p. In conclusion, the main objectives of this thesis, efficient expression and purification of both Cht3p and Sap2p was achieved and completed. Although further studies optimizing the encapsulation conditions for each protein are still needed, this work demonstrates that Cht3p can be successfully encapsulated in liposomal nanoparticles. |
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| Autores principais: | Barbosa, Augusto Alexandre Costa |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | português |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Candida is a genus of ubiquitous yeasts commonly found in healthy individuals and normally acting as harmless commensals in their hosts. However, disruption of the immune defenses can lead to an outgrowth of these yeasts and give rise to local or systemic infections. In fact, Candida, mainly C. albicans, is currently responsible for one of the most common causes of fungal infections worldwide, in part due to an increased development of resistance against the few available therapies. Thus there is an urgent need for the elaboration of new therapies, both for prevention and treatment, among which vaccines may have an important role. Recent studies investigating C. albicans cell wall surface proteins (CWSP) as antigens with DODAB:MO liposomes as carriers and adjuvants led to the detection of a chitinase, Cht3p, as an immune response inducer in mice. Nevertheless, the use of one antigen alone can lead to an evasion of the immune system by C. albicans and hence combination of Cht3p with another antigen, such as the previously studied C. albicans protease Sap2p, is needed. In this study, we successfully developed efficient production and purification protocols for the heterologous production of these two C. albicans proteins. Cht3p could not be produced in Escherichia coli but was successfully produced in a glycosylated and active form in Pichia pastoris. Extracellular production in this host allowed a simplified purification involving ammonium sulfate precipitation and dialysis. Sap2p was successfully produced in both the inactive pro-peptide and active forms in E. coli, with highest production for the former. A simplified, reduced cost, non-chromatographic approach for Sap2p purification, which took advantage of the proteins’ low pH stability, was developed and enabled the obtainment of almost 20 times more purified protein than that previously reported. Preliminary studies investigating various liposomal formulations and conditions previously described and optimized for CWSP (Carneiro et al., 2015) were carried out for both proteins. Cht3p was successfully encapsulated while process optimization is needed for both Sap2p alone and in combination with Cht3p. In conclusion, the main objectives of this thesis, efficient expression and purification of both Cht3p and Sap2p was achieved and completed. Although further studies optimizing the encapsulation conditions for each protein are still needed, this work demonstrates that Cht3p can be successfully encapsulated in liposomal nanoparticles. |
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